Confocal Raman Microscopy
Raman microscopy is a non-contact, non-destructive technique that requires little to no sample preparation.
Figure 1 depicts a general layout for Raman microscopy not including the spectrograph. Raman utilizes a laser (a.) as a source due to weak Raman scattering, about a million times weaker than optical spectroscopy. The beam passes through a pinhole aperture (b.) which filters scattered light, giving higher spatial resolution in the x, y, and z directions. This is the key to confocal microscopy and will be discussed at length later. A beamsplitter (c.) splits the light, with the beam path going to the sample (e.) through the objective (d.). The beam is then split between the camera (f.) and the spectrograph (h.) after going through a rejection filter.
The spot size for a microscope measurement is dependent on three variables: refractive index of the sample (n), the numerical aperture of the objective (NA) and the wavelength of the incident light (see Figure 2). The estimation of the beam waist, or focal plane, volume does not include contributions from scattered light to or from the sample. The advantage of confocal configuration is that it removes the scattered light, bringing the actual beam waist closer to the theoretical size. The apertures also remove the scattered light above and below the beam waist, making depth profiling possible.
Samples can be measured with a spatial resolution as small as 1 μm, and depth profiling can also be easily performed on transparent samples. Using confocal microscopy, measurement can be made in the Z direction for transparent samples, providing a three-dimensional chemical image of a sample. There are several important factors to consider when performing Raman measurements, which have a significant bearing on the quality of the data that can be obtained. Here, we will provide some insights into how to perform good Raman measurements, what may be expected from the instrument, and the key elements that affect performance.
Raman imaging is a powerful technique that provides 3-D spatial information and chemical identification, as seen in the figure below. Samples with dimensions of micrometers to millimeters can be analyzed in just a few minutes. JASCO has developed a technology called QRI that increases the data acquisition speed by up to 50 times compared with conventional mapping, and also offers a dramatic improvement in sensitivity.
Expointing the Raman Effect in Raman and Confocal Raman Imaging Microscopy
The Raman effect takes advantage of the vibrational bond energy in a molecule. Bond energies characterize the nature and environment of atomic interactions in the molecule. A Raman spectrum is a composite of the vibrational frequencies of the various functional groups that may be present in a molecule.
What is Measured in in Raman and Confocal Raman Imaging Microscopy?
Raman spectroscopy and Raman microscopy and especially imaging microscope uses the measured spectrum to identify what material is present in the sample matrix. This can be done using individual peaks at signature frequencies that differentiate the individual components that comprise the sample. Or the entire spectrum can be analyzed chemometrically using algorithms such as PLS, PCA or preferable MCR.
There are a several important considerations in Raman imaging
1) Do you need to analyze the sample in 2D or 3D, for three dimensions, confocal imaging microscopy is required, with the sample stage being moved in XY and Z directions. In this case the optical configuration of objective magnification and numerical aperture (NA), confocal aperture, laser wavelength and spectrograph all play a key role in spatial and spectral resolution.
2) Surface topography – the focal point at the sample surface is key to obtaining the best signal to noise and resulting spectrum. If the sample surface is uneven, rough or the sample relative to the objective lens. With increased magnification the depth of field decreases making this more challenging at higher spatial resolution.
Speed is of the Essence
With large samples to be measured often combined with high spatial resolution, especially when imaging in three dimension, large number of data points are measured with huge volumes of spectra. These have to be combined into both visual images taken through the observation camera and then overlaid with the imaging data. This is a detailed and time-consuming procedure, which can be speeded up by using high throughput microscopes and powerful computer imaging algorithms.